Small-Molecule Inhibitors of SETD8 with Cellular Activity

Abstract

SETD8/SET8/Pr-SET7/KMT5A is the sole protein lysine methyltransferase (PKMT) known to monomethylate lysine 20 of histone H4 in vivo. SETD8’s methyltransferase activity has been implicated in many essential cellular processes including DNA replication, DNA damage response, transcription modulation, and cell cycle regulation. Developing SETD8 inhibitors with cellular activity is a key step toward elucidating the diverse roles of SETD8 via convenient pharmacological perturbation. From the hits of a prior high throughput screen (HTS), SPS8I1–3 (NSC663284, BVT948, and ryuvidine) were validated as potent SETD8 inhibitors. These compounds contain different structural motifs and inhibit SETD8 via distinct modes. More importantly, these compounds show cellular activity by suppressing the H4K20me1 mark of SETD8 and recapitulate characteristic S/G2/M-phase cell cycle defects as observed for RNAi-mediated SETD8 knockdown. The commonality of SPS8I1–3 against SETD8, together with their distinct structures and mechanisms for SETD8 inhibition, argues for the collective application of these compounds as SETD8 inhibitors.

Epigenetic siRNA and Chemical Screens Identify SETD8 Inhibition as a Therapeutic Strategy for p53 Activation in High-Risk Neuroblastoma

“…The IVTI of UNC0379 in NB cells was highly significant when compared with normal cells, suggesting that its characteristics warrant further development. The recent elucidation of structure-activity relationships among SETD8 inhibitors (Ma et al, 2014b) and the identification of other small-molecule inhibitors of SETD8 (Blum et al, 2014) should lead to compounds with increased activity and selectivity (A. Ma unpublished data). As with many targeted compounds, combination studies may prove more efficacious and further studies of inhibitors of SETD8 with Nutlin-3 or DNA damaging conventional cytotoxic agents will be explored.…”

Structure-Based Design of a Covalent Inhibitor of the SET Domain-Containing Protein 8 (SETD8) Lysine Methyltransferase

“…22 A set of quinone compounds were reported as cell-active inhibitors of SETD8. 23 The quinones covalently modified one or more residues on SETD8. Given that the quinone moiety is redox-active and that quinones are known to be promiscuous assay-interference compounds, 24 these compounds may have off-target activities.…”

Progress in the Development of Lysine Methyltransferase SETD8 Inhibitors

“…The inhibitory activity was confirmed in as eries of orthogonal biochemical andb iophysical assays. [33] Dose-response curves determined by as econdary radiometricf ilter paper assay confirmedt he three compounds SPS8I1-3 (small-molecule pool of SETD8 inhibitor,F igure 9; SPS8I1, also known as NSC663284; SPS8I2, also as known as BVT948;S PS8I3,k nown as ryuvidine) as potent inhibitors of SETD8, with apparent IC 50 values of 0.21 AE 0.03, 0.50 AE 0.20, and 0.70 AE 0.20 mm,r espectively.T he selectivities of these compounds were evaluated against ap anel of representative human methyltransferases, including six KMTs( SETD2, GLP,G 9a, SETD8, SMYD2, and SETD7) and three protein arginine methyltransferases (CARM1, PRMT1,a nd PRMT3). Jin and co-workers also synthesized af ew analogues of UNC0379 and studied the structure-activity relationships of the quinazoline scaffold.…”

“…Mechanism of action (MOA) studies, performed by investigating SETD8 inhibition with varying concentrationso f the H4 peptides ubstrate or the cofactor SAM, indicated that UNC0379 was competitive with the peptidesubstrate and noncompetitive with the cofactor.M oreover,s electivity assays showed that the inhibitor was selectivef or SETD8 over 15 other methyltransferases, including G9a and GLP,t he two KMTs that are effectively inhibited by quinazolines. [33] All three inhibitors contained aM ichael acceptor quinonic motif (indicated in red in Figure 9) that could react with active cysteiner esidues.T herefore, the authors performed further mechanistic studies that showed that SPS8I1-3i nhibited SETD8 through an irreversible slow-onset process. [32] They found that the terminal pyrrolidineg roup at the 4position wasw ell tolerated, whereas modifications of length and/ornature of the 5-carbonlinker weredetrimental;m odifications at the 2-position were generally not tolerated, with the only exceptiono ft he replacement of thep yrrolidine with ad imethylamino group;am ethoxy or ethoxy group was required at the 6-position, and replacement with an hydrogen or with al arger group or al ess electron-donating group was disfavored;m odifications at the 7-position could be well tolerateda nd could, eventually, lead to more potent SETD8 inhibitors.…”